Lysosomal storage disorders are a group of more than 40 recessive genetic diseases resulting in deficiencies in lysosomal acid hydrolases (Wraith, J. E., (2001) Dev. Med. Child. Neurol. 43: 639-646). Although individually rare, lysosomal storage disorders have a prevalence of 1 per 7700 live births. Such diseases include Gaucher's disease, Fabry disease, Niemann-Pick disease, mucopolysaccharidoses Type I through VII, Tay-Sachs disease, among many others. Loss of lysosomal enzyme activity results in the progressive accumulation of undegraded substrate within the lysosomes, resulting in engorgement of the organelle, subsequent cellular, tissue, and organ dysfunction, and often death. Lysosomal storage diseases affect multiple organ systems, many of them before birth, resulting in irreversible defects. Clinical treatments for metabolic storage disorders are limited to bone marrow transplantation and enzyme replacement therapy (Cheng, S. H. and Smith, A. E. (2003) Gene Ther. 10: 1275-1281).
Enzyme replacement therapy involves administration of functional lysosomal enzymes to patients. Following administration, the replacement enzymes are secreted by the liver into systemic circulation. Both adjacent and distant cells recapture the secreted enzymes, primarily through the mannose-6-phosphate receptor, which is present on the surface of virtually all cells (Suzuki, K. Lysosomal diseases. In: Graham, D. I., Lantos, P. K. (eds) Greenfield's Neuropathology. Arnold: London, (2002) pp. 653-735). Localized administration of enzyme can replenish at least part of the enzyme population in deficient cells. However, these enzymes generally have short circulating and intracellular half-lives, and therapy requires regular parenteral administration of relatively large amounts of the relevant enzyme.
Enzyme replacement therapy is particularly effective when treating certain lysosomal storage diseases. For example, enzyme replacement in the treatment of Gaucher's and Fabry's disease has been effective in reversing non-neuropathic symptoms of these diseases (Weinreb, N. J. et al, (2002) Am. J. Med. 113: 112-119; Schiffman, R. et al, (2001) JAMA 285: 2743-2749). However, in many lysosomal storage diseases, such as mucopolysaccharidosis type I (i.e., MPS-IH; Hurler's Syndrome), replacement of enzyme can result in potent immunogenic responses against the infused donor proteins. Further, systemically administered enzymes are unable to access sites that arise later in development, such as the CNS and skeletal system. Thus, enzyme replacement therapy is not effective in correcting neurological manifestations and skeletal defects associated with many of these metabolic storage diseases.
Gene targeting has also been used to supply enzymes to patients. However, limited results have been observed thus far. Gene targeting (via targeting vectors, such as lentiviral, retroviral, adenoviral, and adeno-associated viral vectors) can result in efficient delivery to target organs, such as lung, liver, or bone marrow (Marshall, J. et al, (2002) Mol. Ther. 6: 179-189; Du, H. et al, (2002) Gene Ther. 13: 1361-1372). Additionally, intravenous delivery of targeting vectors can result in high-level secretion of the enzymes from the liver and re-uptake in other affected tissues.
However, gene targeting methods also have disadvantages. Expression of the desired enzyme can be transient and decline to basal levels within several weeks. Inflammatory responses mediated by cytotoxic T-lymphocytes against the enzymes have also been observed. Further, correction of more severe defects, such as those residing in the CNS, is unlikely given the difficulties in achieving transfer across the blood-brain barrier, and inducing efficient expression in the quiescent cells of the CNS. In one study, direct intracranial injection using AAV vectors produced only mild correction of cognitive defects in a murine model of mucopolysaccharidosis type VII (MPS VII) (Frisella, W. A. et al., Mol. Ther. (2001) 3: 351-358).
Bone marrow transplantation is another potential therapeutic approach to the treatment of lysosomal storage disorders. Bone marrow transplantation reconstitutes a patient's hematopoietic system with stem cells from healthy immunocompatible donors to establish life-long sources of enzyme (Steward, C. G. (1999) Bone marrow transplantation for genetic diseases. In: Fairbairn, L. J., Testa, N. G. (eds) Blood Cell Biochemistry. Volume 8: Hematopoiesis and Gene Therapy New York: Klewer Academic/Plenum Publishers. p. 13-56). Hundreds of patients with lysosomal storage disorders have been treated by transplantation for many years. For example, it has been reported that treatment of non-neuropathic Gaucher's disease by bone marrow transplantation resulted in almost a complete reversal of symptoms (Hoogerbrugge, P. M. et al, (1995) Lancet 345: 1398-1402).
Bone marrow transplantation has a more limited effect in the treatment of other lysosomal storage disorders, such as MPS-IH. Bone marrow transplantation can reverse some, but not all of the deleterious effects of the disease (Hoogerbrugge, P. M. et al, supra). If not treated by transplantation, patients will continue to deteriorate due to heparin sulfate and dermatan sulfate glycosaminoglycan accumulation in the cornea, central nervous system, liver, spleen, lungs, heart, muscles, tendons, and bones. The degree of neurological improvement is related to the age of the patient, developmental quotient at the time of transplant, and levels of enzyme obtained following transplantation (Peters, C., et al, (1996) Blood 87: 4894-4902; Peters, C. et al, (1998) Blood 91: 2601-8). Younger transplant recipients experience clearing of visceral organs, typically with stabilization or slower deterioration of the CNS. Unfortunately, the skeletal system and many CNS defects remain unaffected following transplant.
Results from bone marrow transplantation and enzyme replacement studies demonstrate that most defects, but not all, can be corrected by supplying the patient with enzyme. However, the failure to correct skeletal and CNS defects in successfully engrafted patients indicates that either there is an insufficient concentration of lysosomal enzymes at the required site (i.e. bone or CNS), or the damage is irreversible. MPS-IH patients typically have <0.13% of normal enzyme levels. However, in patients that are heterozygous for α-L-iduronidase, only 3% of enzyme levels are necessary to yield clinically unaffected leukocytes and fibroblasts (Scott, H. S., et al, (1995) Hum. Mutat. 6: 288-302). This suggests that only low levels of replacement enzyme are necessary and that failure to achieve correction of skeletal and CNS manifestations is due to exceedingly low levels of available enzyme in these sites.
Supplying replacement enzymes early in development may result in greater correction of CNS and skeletal abnormalities. Intrauterine transfer, also known as in utero transplantation, offers the earliest opportunity for optimizing correction of irreversible defects that occur during gestation (Flake, A. W. and Zanjani, E. D., (1999) Blood 94: 2179-2191). The early gestational fetus is immunologically immature and tolerant to foreign antigen, allowing acceptance of allogeneic or xenogeneic cells without the need for immunosuppression. Therefore, immunologic or metabolic reconstitution is possible before birth.
Although patients with severe immune deficiency disorders can be corrected by intrauterine transfer, those with lysosomal storage disorders have not seemingly benefited from this procedure. Only two patients with MPS-IH treated by IUT have been reported (Donahue, J., and Carrier, E., (2002) Cancer Treat Res 110: 177-211). A difference between the results of intrauterine transfer for immune deficiency compared to storage disorders relates to the fact that immunocompetent donor cells (i.e., lymphoid cells) will have a marked competitive proliferative advantage in recipients with immune deficiency disorders. Donor cells (i.e., hematopoietic stem cells (HSCs) or bone marrow mononuclear cells (BMMNCs)) expressing the lysosomal enzyme do not have a proliferative or survival advantage over cells lacking the enzyme.
Animal models of lysosomal storage disorders have confirmed this observation. In a study of MPS VII mice, intrauterine transfer of either syngeneic fetal liver hematopoietic stem cells marked by retroviral vectors, or allogeneic donor cells constitutively overexpressing a human β-glucuronidase (“gus”) transgene resulted in only 0.1% engraftment (Casal, M. L., et al, (2001) Blood 97: 1625-1634). Enrichment of stem and progenitor cells resulted in significantly higher gus activity at 2 months of age, which delayed the onset of manifestations of the disease, suggesting that expression of the enzyme early in fetal life may slow the progression of the disease. Intrauterine transfer of hematopoietic stem cells or donor fetal liver cells does alleviate GAG storage in tissues, such as cortical neurons and glia (Barker, J. E. et al, (2001) Blood Cells Mol. Dis. 27(5): 861-873), as well as in liver (Casal, M. L. (2000) Pediatr. Res. 47(6): 750-756).
The canine model of MPS-IH deficiency was also used to evaluate the therapeutic potential of hematopoietic stem cells infused by intrauterine transfer (Lutzko, C., et al, (1999) Hum. Gene Ther. 10: 1521-1532). Autologous marrow cells were genetically modified to express α-L-iduronidase and transferred in utero into preimmune fetal pups. Neither α-L-iduronidase enzyme nor proviral-specific transcripts were detected in blood or marrow leukocytes of any MPS-IH dog. However, the transduced hematopoietic progenitors could engraft in fetal recipients, contribute to hematopoiesis, and induce immunologic nonresponsiveness to α-L-iduronidase. The therapeutic potential of hematopoietic stem cell gene transfer in dogs appeared to be limited by poor maintenance of proviral α-L-iduronidase gene expression and low levels of genetically corrected circulating leukocytes.
Accordingly, a need exists in the art for improved methods for the treatment of lysosomal storage disorders.